The overall long-term goal of this project continues to be directed at understanding the involvement of zinc-binding proteins in the absorption, metabolism, and biological functions of zinc. Past research has described the hormonal and cytokine regulation of zinc metabolism, concurrent changes in metallothionein gene expression, the first assays of metallothionein mRNA levels in human subjects, and changes in expression as a function of zinc intake. This research has also provided an initial global analysis of zinc-regulated genes, and studies on the integrative aspects of zinc transporter proteins as a function of dietary zinc intake and the physiological factors that influence zinc transporter expression including cytokines, nitric oxide and glucocorticoid hormone. As a result of experiments conducted for this project period, siRNA knockdown of individual transporters responding to physiological mediators showed these proteins influence the intracellular zinc concentrations in cell-specific and organelle-specific modes. Research findings that have been of particular significance have been accomplished during the most recent period of support. The zinc transporter ZIP10 was identified as zinc responsive and is a constituent of the red cell membrane. ZIP14 was identified as a zinc and iron transporter suggesting a potential role of this transporter in iron overload disorders. Zip14 was also identified as IL-6 regulated. ZIP14 was also identified as being regulated by IL-12 by a mechanism that involves nitric oxide as an activator of the Zip14 promoter. Zinc supplementation of human subjects augmented the activation response of T-cells. The mechanism was identified as a ZIP8-mediated zinc efflux from lysosomes with subsequent potentiation of CREB-phosphorylation which enhances IFN3 transcription and IFN3 release. Zinc transport in pancreatic acinar cells was found to be glucocorticoid regulated via increased ZnT2 production and localization to the zymogen granule. A regulatory mechanism involving glucocorticoid receptor and STAT5 was identified. The experiments proposed for the next project period are designed to build on past research on zinc metabolism and function. There remains a major gap between traditional nutritional experiments on zinc, both basic and clinical, and reductionist approaches, which are focused at the molecular level. This project uses an integrative approach directed at reducing the gap in our understanding of zinc nutrition / function between the molecular to organismal levels. The specific aims of the next project period are to: I. Expand our understanding of zinc transporter regulation as related to function;II. Characterize the physiological implications of a ZIP11 knockout mouse III. Examine how zinc transporter proteins respond to zinc intake and physiologic mediators and thus influence intracellular functions of zinc.